Bacillus licheniformis NRRL B-18962 capable of producing levan sucrase in the absence of sucrose

ABSTRACT

The invention relates to an acid stable levan sucrase derived from Bacillus licheniformis NRRL B-19862. The levan sucrase is produced by culturing Bacillus licheniformis NRRL B-19862 in a suitable nutrient medium but in the absence of sucrose. The invention also discloses compositions containing the levan sucrase.

This is a divisional of application Ser. No. 07/884,183, filed May 18,1992, now U.S. Pat. No. 5,334,524.

The present invention relates to an acid stable levan sucrase enzyme,microorganisms producing it, a process for the preparation of this acidstable levan sucrase enzyme and compositions containing it.

Levan sucrase enzymes catalyze the transfer of fructose. More preciselylevan sucrase enzymes (E.C.2.4.1.10) catalyze the transfer of fructosylresidues from sucrose, raffinose or stachyose to an appropriateco-substrate, producing polymers, generally called levans, containingvarying amounts of fructosyl residues consisting of β2→6 linkages.Levans have widespread applications in the chemical and food industry aswell as medicine and research.

Levan sucrase enzymes have been isolated from a variety of microbialsources, microorganisms such as Acetobacter suboxydans, Actinomycesviscosus, Aerobacter levanicum, Bacillus amyloliquefaciens, Bacilluslicheniformis, Bacillus mesentericus, Bacillus subtilis, Glucobacteroxydans, Streptococcus mutans, Streptococcus salivarius, Streptomycesgriseus, Zymomonas mobilis. In most cases, the enzyme is extracellularand heat labile. One of the most important features in the production oflevan sucrase enzymes is that at least 5% sucrose must be added to thegrowth medium in order to induce the synthesis of the enzyme using knownlevan sucrase enzymes producing microorganisms.

As a result, it becomes difficult to separate levan sucrase enzyme fromthe culture broth because the viscosity of the culture broth increasesas levan is produced during fermentation. So there is always a constantsearch for microorganisms requiring low levels or no sucrose for theproduction of levan sucrase.

Japanese Patent Application JP-A-52-82781 discloses a method forproducing an extracellular levan sucrase enzyme in the presence of lowconcentrations of sucrose (0.3% weight/volume) using Bacilluslicheniformis strain AJ 3982 (Institute of Microbial Engineeringdeposition No. 3373). However this enzyme still needs sucrose to beinduced and produced. Moreover this levan sucrase enzyme does notdevelop any enzymatic activity at a pH of 4.0 when held at a temperatureof 55° C. At said temperature it develops only 50% of its maximumactivity at a pH of about 5.2 and about 80% of its maximum activity at apH from about 6 to about 7.8. This narrow pH range restricts the fieldof application of this levan sucrase enzyme. Moreover acid stability ofthe levan sucrase enzyme is very important for widespread commercialapplication of the enzyme.

The present invention aims to provide a levan sucrase enzyme, whichdevelops an enzymatic activity in a wider pH range than the knownenzymes and which is stable at acid pHs at which known enzymes are not.

For this purpose, the present invention provides an acid stable levansucrase enzyme derived from a microorganism belonging to the speciesBacillus and developing, at a temperature of about 55° C. and at a pH of4.0, an enzymatic activity of at least 50% of the maximum activitymeasured at 55° C.

The levan sucrase enzyme according to the invention develops anappreciable enzymatic activity in a wide pH range in the presence of asubstrate such as sucrose. It develops at a pH in the range of about 5.5through about 6.3 a maximum enzymatic activity measured at about 55° C.

More precisely it develops, at a temperature of about 55° C. and at a pHin the range of about 4.0 through about 8.4, an enzymatic activity of atleast 50% of the maximum activity measured at 55° C. It develops, atsaid temperature of about 55° C. and at a pH in the range of about 4.2through about 8.0, an enzymatic activity of at least 70% of the maximumactivity measured at 55° C. It develops, at said temperature and at a pHin the range of about 4.5 through about 7.8, an enzymatic activity of atleast 80% of the maximum activity measured at 55° C. It develops, atabout 55° C. and at a pH in the range of about 4.7 through about 7.2, anenzymatic activity of at least 90% of the maximum activity measured at55° C.

The levan sucrase enzyme according to the invention has an appreciablethermal stability in the presence of a substrate such as sucrose. Itdevelops at a temperature in the range of about 55° C. through about 64°C. a maximum enzymatic activity measured at a pH of 5.5.

More precisely it develops, at a pH of about 5.5 and at a temperature inthe range of about 35° C. through about 75° C., an enzymatic activity ofat least 50% of the maximum activity measured at a pH of 5.5. Itdevelops, at said pH of about 5.5 and at a temperature in the range ofabout 45° C. through about 68° C., an enzymatic activity of at least 80%of the maximum activity measured at a pH of 5.5. It develops at said pHof about 5.5 and at a temperature in the range of about 50° C. throughabout 67° C., an enzymatic activity of at least 90% of the maximumactivity measured at a pH of 5.5.

The levan sucrase enzyme according to the invention is a cell boundenzyme. It has a fructosyl transferase activity and so is able toproduce fructosyl polymers from sucrose consisting of β-D(2→6) linkedfructose which belong to the "levan" family.

The levan sucrase enzyme according to the invention is not a sucroseinducible enzyme, i.e. it does not need sucrose to be induced andproduced.

The present invention also provides a process for the production of anacid stable levan sucrase enzyme using a microorganism in the absence ofadded sucrose to the nutrient medium.

The process according to the invention for the production of an acidstable levan sucrase enzyme comprises the following steps of:

(i) cultivating a microorganism belonging to the genus Bacillus in asuitable nutrient medium by forming a fermentation broth comprising abiomass and

(ii) recovering the levan sucrase enzyme from said fermentation broth,

wherein the microorganism is cultivated in the absence of sucrose.

The present invention also aims to provide a new microorganism belongingto the genus Bacillus producing an acid stable levan sucrase enzyme inthe absence of sucrose in the nutrient medium.

For this purpose the present invention provides a new microorganism ofthe species Bacillus licheniformis producing an acid stable levansucrase enzyme in the absence of sucrose in the nutrient medium. Thepreferred microorganism of the species Bacillus licheniformis has beendeposited with the Agricultural Research Culture Collection (NRRL)Peoria, Ill. under the Budapest Treaty.

The deposit number is NRRL B-18962.

Natural and artificial mutants and derivatives obtained by naturalmodifications or by genetic modifications of the microorganism of thespecies Bacillus licheniformis APMC 84 are also encompassed by thepresent invention.

The levan sucrase enzyme of the present invention can be produced notonly by the strain of Bacillus licheniformis APMC 84 but also by naturalor artificial mutants and other derivatives of this microorganism. Suchmutants can be obtained by well-known techniques, such as X-ray,ultraviolet irradiation, chemical mutagens and genetic engineering.

The levan sucrase enzyme produced in accordance with the process of theinvention is prepared by cultivating the microorganism belonging to thegenus Bacillus in a nutrient medium containing carbon, nitrogen andinorganic salts under aerobic conditions in the absence of sucrose andthen recovering the levan sucrase enzyme therefrom.

The conditions of culture of these microorganisms, such as components ofthe nutrient medium, parameters, temperature, pH, agitation, aeration,are apparent for a man skilled in the art.

Suitable carbon sources include glucose, fructose, maltodextrins,glycerol, corn syrup, starch, hydrolyzed starch or mixtures of two ormore of these carbon sources. Preferred carbon sources are corn syrupand hydrolyzed starch.

Nitrogen sources which can be used include soybean flour, corn steepliquor, potato meal, cottonseed meal, fish meal, yeast, yeast extract ormixtures of two or more of these nitrogen sources. Preferred nitrogensources are soybean flour, cottonseed meal, yeast extract and a mixtureof soybean flour and yeast extract.

Suitable salts include potassium sulfate, manganese sulfate, ammoniumsulfate, ammonium citrate, potassium phosphate, sodium monohydrogenphosphate, sodium dihydrogen phosphate, calcium chloride, sodium titrateor blends of two or more of these salts. Preferred salts include a blendof sodium monohydrogen phosphate, sodium dihydrogen phosphate, calciumchloride and sodium titrate.

The medium containing the above components is sterilized in aconventional manner and inoculated with the appropriate strain ofmicroorganism, preferably with the strain of B. licheniformis and morepreferably the strain of B. licheniformis APMC 84.

Cultivation is conducted aerobically with shaking or under aeratedagitation, typically at a temperature between about 25° and 46° C. forabout 6 to 40 hours and at a pH between about 5.0 and about 8.5.Preferably cultivation is conducted at a temperature between about 30°and 42° C. for about 12 to 30 hours and at a pH between about 6.0 and8.0. Good results are obtained when cultivation is conducted at atemperature between about 36° and 40° C. at a pH between about 6.5 and8.5.

After the cultivation, a fermentation broth comprising a biomass isobtained. This biomass, comprising the cells of the microorganisms withthe cell bound levan sucrase enzyme, is separated and recovered from theculture filtrate using conventional methods such as centrifugation,salting out, precipitation, filtration, ultrafiltration, filtration,microfiltration, centrifugation followed by ultrafiltration orfiltration followed by microfiltration.

The separated and recovered biomass is subsequently washed, preferablyseveral times with water, and more preferably with deionized distilledwater, and homogenized. Repeated washing of the biomass has no effect onthe cell bound levan sucrase enzyme activity leading to the conclusionthat the enzyme is tightly bound to the cell.

The levan sucrase enzyme can further be purified if necessary andaccording to the planned uses. Solubilization of the cell bound enzymecan be obtained in the presence of ionic detergents, such as sodiumcholate or sodium dodecylsulfate (0.25% to 2.5%) or salts solutions,such as Mg⁺⁺ solution. The enzyme can also be precipitated by ammoniumsulfate in the range of 65-95% of saturation at a temperature of about0° C. A sonication technique can also be used to separate the levansucrase enzyme and the cells, if necessary.

The levan sucrase enzyme can be formulated into compositions containingthe levan sucrase enzyme usable in various industries, such as inparticular food industries, pharmaceutical industries and chemicalindustries.

The levan sucrase enzyme is formulated according to its plannedapplications. Usually stabilizers and preservatives are also added tothe enzyme compositions. For example enzyme can be stabilized by addingglycerol (50 volume %), ammonium sulfate (3.2 mol/l) or sodium chloride(3 mol/l) to the aqueous solution of the enzyme.

For medical applications the enzyme may be used preferably inlyophilized form.

For food applications the enzyme may be used immobilized by physical orchemical coupling of the enzyme to essentially insoluble inert carriermaterials which facilitate their use in flow-through reactors. Usuallythe enzyme is attached to the carrier. Materials utilized for thecarrier include organic and inorganic materials, such as porous granulardiatomaceous earth treated with a solution of polyamine andglutaraldehyde, granular activated carbon, surface-active material suchas alumina, carbon, clay, zirconia, titania, ion exchange resins,cellulose or glass, chemically activated supports like cellulose,agarose, synthetic polymers, gels of for example polyacrylamides, silicaand starch, enzyme being captured within a polymer matrix. Preferablylevan sucrase enzyme comprising Bacillus whole cells may be directlyused in the immobilization process without isolation and purification ofthe enzyme.

The compositions containing the levan sucrase enzyme of the presentinvention can be utilized in either solid or liquid form. Thesecompositions can be made into a final product which is either liquidsolution, solid, granular, powder or a slurry.

The compositions according to the invention can be used for theproduction of levans from sugars containing fructosyl residues, such assucrose, raffinose or stachyose. These sugars can derived from rawmaterials, such as ray sugarbeets, purified juice from crushed orshredded sugar beets, molasses, cane sugars, beet sugars, plant sugars.

Levans obtained can be high molecular weight levans, low molecularweight levans (fructooligosaccharides) and fructosyl polymers.

The present invention is further illustrated by the following examples.

EXAMPLE 1

A seed medium used for inoculum development is prepared with thefollowing components: calcium chloride 0.02% weight/volume, sodiumcitrate 0.3%, hydrolyzed starch 2.6% sold under the trademark MALTRIN100 (GPC), cottonseed meal 4.6% sold under the trademark PHARMAMEDIA(TRADERS PROTEIN), sodium monohydrogen phosphate 0.21% and sodiumdihydrogen phosphate 0.54%. This seed medium is sterilised at 125° C.during 30 minutes in seed flasks which are 250 ml triple baffledErlenmeyer flasks containing 50 ml of seed medium. Then they areinoculated from a frozen glycerol culture of the strains of B.licheniformis APMC 84.

The seed of this strain is developed on a gyratory shaker at 37° C. for24 hours prior to its use as a source of inoculum for the productionflasks.

The medium used for the production of levan sucrase enzyme is preparedwith the following components: corn syrup 10.3% weight/volume sold underthe trademark STALEY 200 corn syrup (A. E. STALEY), soybean flour 5.5%sold under the trademark PROMOSOY 100 (CENTRAL SOYA), yeast extract(sold by UNIVERSAL FOODS CORP) 0.32%, sodium monohydrogen phosphate 0.7%and sodium dihydrogen phosphate 0.7%.

This production medium is sterilised at 125° C. during 30 minutes inproduction flasks which are 250 ml triple baffled Erlenmeyer flaskscontaining 50 ml of production medium.

The inoculum size amount is 2% volume/volume. The inoculated productionflasks are incubated on a gyratory shaker at a temperature of 37° C. for16 to 20 hours.

An increasing of the viscosity is not observed when levan is produced infermentation broth.

The enzyme produced can be isolated easily with almost no stickiness.

The biomass from the flasks is separated from the fermentation broth bycentrifugation whereupon the cells are washed with deionized-distilledwater two times and homogenized.

The levan sucrase enzyme according to the invention is recovered.

Levan sucrase enzyme unit (LSU) is defined as the amount of enzymeactivity required to produce one micromole of glucose per minute underconditions of the assay.

The enzyme activity was measured using sucrose as substrate. To onemilliliter of an enzyme aliquot, 7.5 ml of 0.01M citrate buffer, pH 5.5containing 60% sucrose (weight/weight) was added. Then the volume of thereaction mixture was adjusted to 10 ml using distilled rarer andincubated at 55° C. for one hour. After the specified time, theenzymatic reaction was terminated by heat at 95° C. for 10 minutes. Theglucose content of the reaction products was determined by highperformance liquid chromatographic (HPLC) analysis (Brobst, K. M., andH. D. Schobell, 1982. Starch/Starke, 34, 117-121).

The evaluation of the effect of pH on the activity of the levan sucraseenzyme was determined by carrying out the enzymatic transfructosylationreaction after incubation at 55° C. during one hour at different pHlevels; i.e., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5., 7.0, 7.5 and 8.0 using anappropriate phosphate buffer (0.01M). The amount of glucose formed wasdetermined using HPLC (High Performance Liquid Chromatographicanalysis). The activity was calculated.

At 55° C., maximum activity is developed at pH around 5.5-6.0.

FIG. 1 illustrates the effect of pH on the activity of the enzyme at atemperature of 55° C. In this figure the abscissae show the pH and theordinates show the activity expressed as percent of the maximum activitydeveloped at pH 5.5 and at 55° C.

The symbol 0 represents the data for the enzyme according to theinvention and for comparison purposes the symbol # represents the datataken from Japanese patent application JP-A-52-82781.

The enzyme of the present invention is more stable in the acidic pHrange than the enzyme disclosed in said prior Japanese patentapplication.

The enzyme of the present invention exhibits at 55° C. and at pH 4.0over 50% of its maximum activity measured at 55° C. whereas the enzymeof the prior art did not show any activity at pH 4.0 and at saidtemperature.

The FIG. 1 shows that the levan sucrase enzyme according to theinvention develops an appreciable enzymatic activity in a wide pH rangein the presence of sucrose. It develops at a pH in the range of about5.5 through about 6.3 a maximum enzymatic activity measured at 55° C.More precisely it develops, at a temperature of about 55° C. and at a pHin the range of about 4.0 through about 8.4, an enzymatic activity of atleast 50% of the maximum activity measured at 55° C. It develops, atsaid temperature of about 55° C. and at a pH in the range of about 4.2through about 8.0, an enzymatic activity of at least 70% of the maximumactivity measured at 55° C. It develops, at said temperature and at a pHin the range of about 4.5 through about 7.8, an enzymatic activity of atleast 80% of the maximum activity measured at 55° C. It develops, atabout 55° C. and at a pH in the range of about 4.7 through about 7.2, anenzymatic activity of at least 90% of the maximum activity measured at55° C.

The evaluation of the effect of temperature on the activity of levansucrase enzyme was determined by carrying out the enzymatic reaction atpH 5.5 after incubation during one hour at different temperatures, i.e.,40° C., 50° C., 55° C., 60° C., 70° C. and 80° C.

The reaction mixture consisted of 7.5 ml of 60% (weight/volume) sucrosesolution in 0.01M citrate-phosphate buffer at a pH of 5.5 and 1.0 ml ofwashed biomass containing 30 LSU/ml enzyme activity.

At pH 5.5 maximum activity is developed at a temperature of about 60° C.

The effect of temperature on levan sucrase enzyme activity is shown inFIG. 2. In this figure the abscissae show the reaction temperature in °C. and the ordinates show the activity expressed as percent of themaximun activity developed at 60° C. and at pH 5.5. The symbol 0represents the data for the enzyme according to the invention and forcomparison purposes the symbol # represents the data taken Japanesepatent application JP-A-52-82781.

The FIG. 2 shows that the levan sucrase enzyme according to theinvention has an appreciable thermal stability in the presence ofsucrose.

It develops at a temperature in the range of about 55° C. through about64° C. a maximum enzymatic activity measured at a pH of about 5.5.

More precisely it develops, at a pH of about 5.5 and at a temperature inthe range of about 35° C. through about 75° C., an enzymatic activity ofat least 50% of the maximum activity measured at a pH of 5.5. Itdevelops, at said pH of about 5.5 and at a temperature in the range ofabout 45° C. through about 68° C., an enzymatic activity of at least 80%of the maximum activity measured at a pH of 5.5. It develops at said pHof about 5.5 and at a temperature in the range of about 50° C. throughabout 67° C., an enzymatic activity of at least 90% of the maximumactivity measured at a pH of 5.5.

EXAMPLE 2

The effect of levan sucrase enzyme concentration (LSU/g sucrose) on thecomposition of the reaction products at different time intervals wasmeasured at a temperature of 55° C. at a pH of 5.5.

In a typical experiment, 4.5 g of sucrose in 9.5 ml of water wasincubated with 0.5 ml aliquot of biomass containing different amounts oflevan sucrase enzyme, i.e., 4.5, 9.0 and 22.5 LSU at a temperature of50° C. Samples were withdrawn at different intervals of time and thereaction was terminated by heating at 90° C. for 10 minutes.

The product's composition was determined by high performance liquidchromatography (Table 1).

                  TABLE 1                                                         ______________________________________                                        Effect of levan sucrase enzyme concentration (LSU/G sucrose)                  on the composition of the reaction products                                   Enzyme                                                                        Concentration                                                                            Time   Composition of the reaction products %                      LSU/g Sucrose                                                                            [hr.]  Fructose Glucose                                                                              Sucrose                                                                              Levan                                ______________________________________                                        1 LSU/G    2      2.48      4.93  86.82   5.77                                           4      4.13      9.63  76.24  10.00                                           6      5.50     41.06  66.74  13.70                                           8      6.94     18.41  58.54  16.56                                           12     7.49     23.60  47.30  21.61                                           16     8.60     28.95  37.90  24.55                                           24     9.79     36.35  23.14  30.71                                           36     10.90    41.57  13.40  34.14                                           44     11.24    42.91  10.55  35.29                                2 LSU/G    2      3.99     10.10  76.55   9.36                                           4      6.41     18.59  58.28  16.72                                           6      7.87     26.09  43.09  22.95                                           8      9.03     32.33  31.59  27.08                                           12     10.22    39.27  17.74  32.77                                           16     10.93    42.93  11.11  35.02                                           24     11.76    45.25   6.90  36.08                                5 LSU/G    2      7.19     23.94  48.02  20.35                                           4      9.76     38.61  19.23  32.39                                           6      10.98    44.28   8.71  36.02                                           8      11.41    45.65    6.48 36.46                                           12     12.34    45.32   6.43  35.91                                           16     13.01    45.46   6.06  35.47                                ______________________________________                                         [hr.] represents the unit of hour.                                            The rate of formation of glucose from sucrose by levan                   

The rate of formation of glucose from sucrose by levan sucrase enzymeincreased with increasing concentration of the enzyme. An amount of 35%levan was produced in all the cases. A amount of 45% glucose wasproduced in all the cases.

EXAMPLE 3

The thermal stability of the enzyme in the presence of substrate is veryimportant for widespread commercial application of the enzyme. Theeffect of temperature on the composition of the reaction products wasdetermined at temperatures of 40° C., 50° C. and 60° C. The reactionmixture consisted of 7.5 ml of 60% (weight/weight) sucrose solution in0.01M citrate-phosphate buffer at a pH of 5.5 and 2.5 ml of enzymesolution containing 40 units LSU. Samples were withdrawn at differentintervals time and the reaction was terminated by heating at 90° C. for10 minutes.

The products composition was determined by high performance liquidchromatography (Table 2).

                  TABLE 2                                                         ______________________________________                                        Effect of temperature on the composition of the reaction products             Reaction  Time   Composition of the Reaction Products %                       Temperature                                                                             [hr.]  Fructose Glucose                                                                              Sucrose                                                                              Levan                                 ______________________________________                                        50° C.                                                                            4      7.50    23.31  47.33  21.86                                            8      9.15    35.54  24.16  31.15                                           14     11.28    47.30   7.27  34.15                                           24     11.09    43.32  10.58  35.01                                 55° C.                                                                            4      7.92    24.32  45.26  22.50                                            8     10.63    35.58  23.39  30.41                                           14     13.80    46.07   9.07  30.93                                           24     14.43    46.77   6.20  32.61                                 60° C.                                                                            4      8.65    23.66  46.35  21.34                                            8     11.51    32.14  29.98  26.36                                           14     15.11    42.87  18.09  23.93                                           24     14.55    40.36  14.74  30.36                                 ______________________________________                                    

The data in Table 2 demonstrate that the enzyme of the present inventionhas good thermal stability in the presence of a substrate between 50°and 65° C. at a pH of 5.5.

EXAMPLE 4

High substrate concentration is always preferred for commercial successof any transformation because of the high cost of the energy in theevaporation of reaction products.

The effect of sucrose concentration on the composition of the reactionproducts was studied at different intervals during incubation of sucroseat a temperature of 55° C. with the enzyme at a pH of 5.5.

Acetate buffer (0.1M) at a pH of 5.5 containing different amounts ofsucrose was prepared. Appropriate amounts of enzyme were added to afinal concentration of 10 LSU/g sucrose and incubated at 55° C.

Samples were withdrawn at different intervals of time and the enzymaticreaction terminated by heating at 90° C. for 10 minutes.

The composition of the reaction products was then determined by HPLC(Table 3).

                                      TABLE 3                                     __________________________________________________________________________    Effect of sucrose concentrations on the composition of the                    reaction products                                                             Sucrose                       Ratio of                                        Concentration                                                                         Time                                                                             % Composition      Levan to Free                                   g/100 ml                                                                              [hr.]                                                                            Fructose                                                                           Glucose                                                                            Sucrose                                                                            Levan                                                                             Fructose                                        __________________________________________________________________________    30       5  9.75                                                                              25.69                                                                              43.88                                                                              21.57                                                                             2.21                                                     8 12.47                                                                              32.78                                                                              30.09                                                                              24.67                                                                             1.98                                                    12 16.14                                                                              43.04                                                                              17.75                                                                              23.08                                                                             1.43                                                    24 17.67                                                                              45.35                                                                               6.66                                                                              30.33                                                                             1.73                                            45       5  8.99                                                                              29.44                                                                              36.84                                                                              24.72                                                                             2.75                                                     8 10.71                                                                              36.40                                                                              22.75                                                                              30.13                                                                             2.81                                                    12 13.66                                                                              44.31                                                                              11.03                                                                              31.00                                                                             2.27                                                    24 13.56                                                                              42.75                                                                              10.20                                                                              33.46                                                                             2.46                                            60       5  6.93                                                                              29.47                                                                              34.12                                                                              29.49                                                                             4.26                                                     8  8.46                                                                              39.93                                                                              20.55                                                                              34.05                                                                             4.02                                                    12 11.12                                                                              44.98                                                                               8.78                                                                              34.12                                                                             3.16                                                    24 14.03                                                                              44.98                                                                               5.37                                                                              35.62                                                                             2.54                                            75       5  6.25                                                                              29.15                                                                              33.34                                                                              31.27                                                                             5.00                                                     8  7.57                                                                              37.30                                                                              19.00                                                                              36.12                                                                             4.77                                                    12  9.45                                                                              44.26                                                                               8.93                                                                              37.34                                                                             3.95                                                    24 10.76                                                                              43.31                                                                               4.96                                                                              41.12                                                                             3.82                                            __________________________________________________________________________

The results set out in Table 3 show that the transfructosylation wasincreased with increasing concentration of sucrose. The ratio of levanproduced to the free fructose at any time interval was higher at higherconcentration of substrate compared to the ratio at lower concentrationof the substrate. For example, the ratio of levan to free fructose liesbetween 1.4 to 2.2 at 30% sucrose concentration, whereas the ratio is4.5-5.0 at 75% sucrose concentration. This strongly suggests that thelow water activity favors the levan production.

We claim:
 1. A biologically pure culture of Bacillus licheniformis NRRLB-18962.
 2. A mutant of the Bacillus licheniformis, is NRRL B-18962wherein said mutant is capable of producing levan sucrase in the absenceof sucrose.